PROJECT SUMMARY/ABSTRACT Substantial evidence supports health benefits associated with aspirin use, particularly for individuals at increased risk for cardiovascular disease (CVD) and colorectal cancer (CRC). In 2016, the U.S. Preventive Services Task Force recommended low-dose (81 mg) aspirin for primary prevention of CVD and CRC despite an incomplete understanding of the biological mechanisms underlying aspirin?s effects on the colon. We have proposed an interrelated framework for aspirin?s biological mechanisms through direct inhibition of prostaglandins within epithelial cells. To expand this framework, we now propose the novel hypothesis supported by compelling preliminary data that the gut microbiome may mediate the biological effects on colorectal mucosa associated with aspirin. The gut microbiome is a key determinant for gut homeostasis and is increasingly implicated in the development of colorectal neoplasia. However, prospective studies are required to define the specific role of the gut microbiome in the development of mucosa at-risk for neoplasia and how microbial dynamics are impacted following intervention with aspirin. The overall goal of this proposal is to interrogate the joint impact of aspirin on colonic epithelial cells and the gut microbiome in humans to refine and establish causal mechanisms, including PG pathways, which will be further validated using novel, in vitro, patient-derived modeling approaches. We hypothesize that by fully interrogating these additional mechanisms an integrated network may be developed that comprehensively informs a precision prevention strategy. To address this, we will leverage biospecimens (colonic biopsies and stool) collected within our randomized, double-blind, placebo- controlled trial of aspirin, ASPIRED. In Aim 1, we will deeply characterize the effects of randomized aspirin treatment on colorectal mucosa through RNA sequencing of epithelial cells collected from mucosal biopsies and on the gut microbiome by performing integrated whole-shotgun metagenomics, metatranscriptomics, and metabolomics to investigate the effect of randomized aspirin treatment on the gut microbiome. In Aim 2, will leverage intestinal organoid cultures, or three-dimensional ?mini-guts?, to culture epithelial cells derived from the same patients with aspirin (direct effects) and/or a priori microbial metabolites (indirect effects) to refine chemopreventive mechanisms. The results will further elucidate a role for the gut microbiome in the health and disease states of the alimentary tract and aligns with the overall mission of the NIDDK. This proposal will also offer a promising young investigator the opportunity to further develop a niche within which to pursue independent lines of inquiry and expand his bioinformatics and translational methodological skillsets. This will be an important first step for the candidate to achieve his goal of leveraging basic science training (pre-doctoral) and experience in clinical gastroenterology trials (post-doctoral) to become an effective bridge between the laboratory and the clinic as an independent academic investigator.